Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9–Cas12a platform

Thomas Gonatopoulos-Pournatzis, Michael Aregger, Kevin R. Brown, Shaghayegh Farhangmehr, Ulrich Braunschweig, Henry N. Ward, Kevin C.H. Ha, Alexander Weiss, Maximilian Billmann, Tanja Durbic, Chad L. Myers, Benjamin J. Blencowe, Jason Moffat

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71 Scopus citations


Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9–Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.

Original languageEnglish (US)
Pages (from-to)638-648
Number of pages11
JournalNature biotechnology
Issue number5
StatePublished - May 1 2020

Bibliographical note

Funding Information:
We thank Q. Morris for advice on machine learning implementation, F. Zhang and B. Zetsche (Broad Institute, MIT) for providing Cas12a reagents and insights, and members of the Moffat, Blencowe and Myers laboratories for helpful discussions. G. O’Hanlon, Q. Huang, C. Sheene and S. Sidhu are gratefully acknowledged for assistance with sequencing and molecular biology experiments. T.G.-P. was supported by postdoctoral fellowships from the European Molecular Biology Organization, Ontario Institute of Regenerative Medicine (OIRM) and Canadian Institutes for Health Research. M.A. was supported by a Swiss National Science Foundation fellowship. H.N.W. was supported by a National Institutes of Health (NIH) Biotechnology Training Grant. This research was funded by grants from Canadian Institutes for Health Research (to B.J.B. and grant no. MOP-142375 to J.M.), Medicine-by-Design Canada First Research Excellence Fund (grant no. CITPA-2016-11 to B.J.B. and J.M.), OIRM (to B.J.B.), Genome Canada (grant no. OGI-157 to J.M.), NIH (to C.L.M.) and the National Science Foundation (to C.L.M.). J.M. is a Canadian Research Chair in Functional Genomics. B.J.B. holds the Banbury Chair of Medical Research at the University of Toronto.


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